Immunogens comprising capsular saccharide antigens conjugated to carrier proteins are well known in the art. Conjugation converts T-independent antigens into T-dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop, and the prototype conjugate vaccine was for Haemophilus influenzae type b (Hib) [e.g see chapter 14 of ref. 1]. Since the Hib vaccine, conjugated saccharide vaccines for protecting against Neisseria meningitidis (meningococcus) and against Streptococcus pneumoniae (pneumococcus) have been developed. Other organisms where conjugate vaccines are of interest are Streptococcus agalactiae (group B streptococcus) [2], Pseudomonas aeruginosa [3] and Staphylococcus aureus [4].
Rather than use full-length capsular saccharides, it is possible to select oligosaccharide fragments of desired size after a hydrolysis step [e.g. ref. 5], and it has been reported that conjugates made with intermediate chain-length oligosaccharides offer improved immunogenicity [e.g. refs. 6 & 7]. Of the three N. meningitidis serogroup C conjugated vaccines that have been approved for human use, Menjugate™ [8] and Meningitec™ are based on oligosaccharides, whereas NeisVac-C™ uses full-length polysaccharide. Measurement of oligosaccharide length (e.g. by measuring the degree of polymerisation, or ‘DP’ i.e. the number of repeating units in the chain) can therefore be used for indirect assessment of immunogenicity.
Where oligosaccharide fragments are included in a vaccine, quality control for manufacturing and release requires that oligosaccharides have a defined length, and that this length is consistent between batches. Thus DP is also useful in quality control, and the European Directorate for Quality of Medicines (EDQM) has an Official Control Authority Batch Release (OCABR) for conjugated Hib vaccines that specifically requires submission of data relating to DP and molecular size distribution of saccharides used during manufacture.
DP can also be used for monitoring vaccine stability. Saccharide antigens can readily depolymerise at ambient temperatures [9,10], causing a decrease in immunogenicity and an increase in vaccine heterogeneity. Such changes can be monitored by following DP over time during storage.
Average DP in an oligosaccharide pool can be measured using a number of methodologies, and in some cases the choice of method will depend on the saccharide under analysis. Techniques such as colorimetric and/or enzymatic analysis have been described for oligosaccharides from Hib and from serogroups A and C of meningococcus [5,11,12], but the inventors have found that the glycosidic linkages in the saccharides of meningococcal serogroups W135 and Y (‘MenW135’ and ‘MenY’) mean that these techniques cannot be used.
Although methods for measuring DP of MenA and MenC saccharides for conjugate vaccines have previously been described [e.g. refs. 5 & 13], there remains a need for methods that can be applied to the saccharides of serogroups W135 and Y. It is thus an object of the invention to provide improvements in methods for DP assessment of saccharides, and in particular to provide methods that can be used to measure DP for saccharides from meningococcal serogroups W135 and Y.